1. Field of the Invention
The present invention relates to the treatment of blood disorders; and in particular, to a method and composition for the treatment of hemoglobinopathies, including .alpha.-thalassemia, .beta.-thalassemia, sickle cell diseases such as sickle cell SC (i.e., HbSC), sickle cell anemia (i.e., HbSS), and sickle cell trait (i.e., HbAS), for example, and combination hemoglobinopathies such as HbSS/.beta.-thalassemia, for example.
Sickle cell disease is an inherited disease wherein the patient carries two abnormal .beta..sup.s genes, at least one of which codes for an abnormal type of hemoglobin molecule hemoglobin S (HbS). The disease itself stems from inadequate oxygen transport by red blood cells due to the presence of HbS. In sickle cell disease, HbS replaces normal hemoglobin, hemoglobin A (HbA), and differs from HbA only in that glutamic acid is substituted for valine at position 6 of the beta chain of the globin molecule. This one variation results in HbS being less soluble than HbA, especially in the reduced state, where it forms long, crystalline masses to cause the red blood cells to distort into the shape of sickles.
HbS is inherited as a mendelian dominant such that there are both homozygous and heterozygous states. The most common and most severe form of the disease is HbSS, the homozygous state, (also referred to as sickle cell anemia). HbSS makes up from 80% to 100% of the total hemoglobin in individuals affected with sickle cell disease. Sickle cell trait carriers (HbAS) are heterozygous for HbS and usually show no sign of the disease. However, since about 25% to 40% of the total hemoglobin in trait carriers is HbS, sickle cell trait carriers risk hemolysis when exposed to low oxygen tension, such as during anesthesia, for example. Statistically, the marriage of two trait carriers results in a 25% chance that one of their children will be afflicted with sickle cell disease (i.e., homozygous) and a 50% chance that their children will have the sickle cell trait (i.e., heterozygous). Other sickle hemoglobinopathies related to HbSS include HbSC, HbSD, and HbSE.
Sickle cell disease is most prevalent in the black races, but it is also known in other races surrounding the Mediterranean and in India. About 30% of Mediterranean people carry the trait (i.e., are carriers). About 1.2 million Afro-Americans carry the sickle cell trait while about another 0.2% have sickle cell disease. The disease, however, is much more prevalent in central Africa, where 40-45% carry the sickle cell trait, while about 4-10% have other hemoglobinopathies. For example, in Gabon, the disease represents 10-30% of out-patient pediatric consultations and 25% of in-patient pediatrics.
The most common manifestation of sickle cell disease, as well as other hemoglobinopathies, is an extremely painful "crisis," typically lasting several days, and affecting one or more local parts of the body. The crisis often occurs following physical stress, and appears to be due to limited oxygen supply to the affected part resulting from an inferior oxygen-carrying capability of HbS as well as to its tendency to aggregate in insoluble gels within the red blood cell to cause the cell to distort into the shape of a sickle. The sickling of the cells results in hemolytic anemia and capillary obstruction due to thrombosis. The resulting hypoxia leads to fatty degeneration in the liver, kidney, and heart. Other organs are also at risk to the effects of hypoxia due to vascular obstruction which may occur at many other sites in the body, as well.
The symptoms of sickle cell disease, usually related to the anemia, normally occur during the second year of life. Symptoms include slight jaundice, fever, and severe bone, joint, and abdominal pain. Children are particularly susceptible to infections such as salmonella osteomyelitis and bacterial meningitis. The prognosis for individuals with sickle cell disease is very poor, and those afflicted with severe forms of the disease usually do not live through their teen years.
Thalassemia is another inherited hemoglobinopathy in which there is a quantitative reduction in either the .alpha.-chains (.alpha.-thalassemia) or .beta.-chains (.beta.-thalassemia) of globin. In .beta.-thalassemia, there is an increase in HbA.sub.2 (.alpha..sub.2 .delta..sub.2) and/or HbF (.alpha..sub.2 .gamma..sub.2), due to the combination of .alpha.-chains, which are in excess, with .delta. and .gamma. globin chains. In normal individuals, HbF is absent and HbA.sub.2 comprises 2-4% of normal hemoglobin. About 30 million persons carry a .beta.-thalassemia gene, and about 8,000-10,000 children are born with .beta.-thalassemia major every year.
In thalassemia, the red blood cells are thinner and more fragile than normal red blood cells. Further, the excess .alpha.-chains present in .beta.-thalassemia also may contribute to hemolysis due to their instability. Thus, the red blood cells in this anemic blood disorder are more susceptible to hemolysis, and therefore have a shortened lifespan.
2. Description of the Related Art
Despite the fact that the cause of sickle cell disease (i.e., the very minor structural variation in the mutant hemoglobin) has been known for many years, little progress has been made in suitable treatment of the disease. Presently, the major treatment for the painful crises is medication for relief of pain, which merely treats the immediate symptoms. Tissue damage, often involving major organs, occurs with each successive episode of oxygen deprivation, and the cumulative effects of the disease are debilitating.
Several attempts have been made in the past to treat and manage sickle cell disease chemotherapeutically, but all have resulted in serious adverse health conditions. Many physiological laws have been applied biochemically in order to achieve success in this regard, but all efforts were frustrated by gross side effects. Examples of such attempts include the transformation of hemoglobin to carboxyhemoglobin (Sirs, J. A. "Preliminary Communication: The Use of Carbon Monoxide to Prevent Sickle-Cell Formation." Lancet, 971-972 (1963)), acetylation of hemoglobin molecules with aspirin; Shamsuddin, et al, "Sites of Acetylation of Sickle-Cell Hemoglobin by Aspirin," Proc. Nat'l Acad. Sci., 71:43 (1975)), cross-linking hemoglobin molecules with dimethyl-adipimidate (Lubin, B. H. et al., "Dimethyl Adipimidate. A New Antisickling Agent." Proc. Nat'l Acad. Sci., 72:43 (1975); Waterman M. R., et al., "Antisickling Nature of Dimethyl Adipidimate" Biochem. Biophys. Res. Commun., 63:580 (1975)), and the use of carbonic anhydrase inhibitors (Hilowitz, G., "Sickle-cell Disease: New Method of Treatment, Preliminary Report," Br. Med. J., 2:266 (1975)).
Other antisickling agents have been employed; however, their disadvantages have by far outweighed their advantages in the treatment of sickle cell disease. Some examples of such agents include urea, cyanate, procaine, pyridoxine, phenothiazines, steroids, nitrogen mustard, and 3,4 dihydro-2,2,-dimethyl-2H-1-benzopyran-6-butyric acid.
Over the years there has been much interest in the anti-sickling effects of roots from the tree Fagara zanthoxyloide, the genus of which Fagara contains over 100 different chemical compounds. In 1975, Sofowora, et al. reported the isolation and in vitro nonsickling characteristics of 2-hydroxymethylbenzoic acid which he and his co-workers extracted from the Fagara zanthoxyloides root. (Sofowora, et al. Lloydia, 38: 169-171 (1975)). In that same year, Honig et al. reported the in vitro effect of the aqueous Fagara extract described by Sofowora et al. above on the oxygen affinity and erythrocyte sickling in samples of whole blood from sickle cell patients. (Honig et al., Lloydia, 38:387-390 (1975).
In 1977, Fadulu, the inventor of the present patent application, reported the in vitro anti-sickling effect of an extract of the Fagara zanthoxyloides root obtained from extraction with benzene, chloroform, ethyl acetate, and ethyl alcohol (Fadulu, Faculty Research Journal, 20-31 (1977). The chemical characteristics of the extract described in Fadulu's article suggested the presence of compounds similar in activity and structure to naturally occurring anesthetics. It was also observed from the IR spectrum that the extracted compound contained free sulfhydryl groups which were further hypothesized to play a role in its anti-sickling activity.
In addition to the Fagara root, other plants have been reported to contain compounds having in vitro antisickling properties. U.S. Pat. No. 4,473,559 to Robinson, for example, suggests in vitro anti-sickling properties of a mixture of compounds derived from porphyrinic or chlorophyllic compositions. U.S. Pat. No. 3,102,891 to Allen is incorporated by reference in Robinson and describes the extraction process for isolating the foregoing compound.
Other compounds have been reported to affect the levels of fetal hemoglobin (HbF), which has been found to ameliorate the clinical course of sickle cell disease. The first attempt to manipulate this change was by the use of 5-azacytidine, a cytotoxic agent. Administration of this agent increased HbF levels in sickle cell patients. Other cytotoxic agents such as cytosine arabinose, hydroxyurea, and hematopoietic factor erythropoietin have demonstrated similar effects. In a recent clinical trial, Perrine et al. reported rapid stimulation of the HbF following the administration of arginine butyrate (N. Engl. J. Med., 328:81 (1993)). However, while these drugs that increase HbF hold promise in the therapy of sickle cell disease, clinical response to these drugs has not been impressive. Thus, a potentially effective drug for treating sickle cell disease or thalassemia would normalize the concentration of HbF to therapeutic levels.
Based on current knowledge of sickle cell disease, it appears feasible to develop a drug which will alleviate all of the symptoms of the disease and provide perfectly normal lives and life-expectancies for sickle cell patients. This drug would not cure the disease (because it is genetic in origin), but should effectively treat the disease by alleviation or prevention of its symptoms. The requisite capabilities of a potentially useful drug have been defined by the Sickle Cell Disease Branch of the United States National Institutes of Health (NIH). The requirements are specified in terms of several laboratory bioassays providing seven parameters that a candidate drug must meet.
Many drugs are known which are effective in normalizing one or more of these parameters, and fifteen of the most promising ones have been evaluated thoroughly (in vitro) by R. L. Nagel's group (Blood, 61:693 (1983)). However, as shown by Nagel, no one drug is capable of normalizing all seven parameters. Nagel's retrospective study thus explains the prior clinical failures of these drugs.
The extract of the present invention, however, displayed excellent responses in all these bioassays, as can be seen in the examples below. The effectiveness of the inventive extract in all seven U.S. NIH tests sets it apart as the only known extract which fulfills all of the in vitro requirements judged to be essential for a truly effective anti-sickling drug.